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Effect of Hofmeister series salts and BSA on fluoresceincompounds

Swathi Korrapati1,2, Shilpa Kammaradi Sanjeeva1, U. Vijayalakshmi2,Phani Kumar Pullela1,2,3. *

1Bigtec Pvt Ltd., 59th “C” cross, 4th “M” Block, Rajajinagar, Bangalore, India - 5600102School of Advanced Sciences, VIT University, Vellore, Tamil Nadu, India - 632014

3CMR Institute of Technology, ITPL Main Road, Bangalore, Karnataka, India – 560037

Abstract: The biophysical parameters like pH of the medium, temperature, refractive index,extinction coefficient of fluorescent compound etc. are responsible for fluorescence quantumyield. Even after maintaining these parameters constant in an assay, peculiarities do occurwith respect to quantum yield due to presence of salts and proteins present in the medium.Most of the fluorescent compounds have different quantum yields in presence of Hofmeisterseries salts and their behaviour has never been studied thoroughly. We performed asystematic study of effects of Hofmeister series salts (urea, sodium chloride, ammoniumsulphate, and guanidine isothiocyanate) and BSA (0.1 mg/mL) on fluorescence quantum yieldfor seven different fluorescent compounds (fluorescein, fluorescein isothiocyanate, DSSA,FITC-attached staurosporine, FITC-attached estrogen, FITC-attached NADH and FITC-attached NADPH). Presence of guanidine isothiocyanate and (NH4)2SO4) has drasticallyreduced fluorescence quantum yield of all compounds. In general, presence of salts like ureahas increased quantum yield, BSA and other salts have minimum effect on quantum yield.Among the dyes studied, the two groups obtained are fluorescein, FITC and E2-FITC in thefirst; FITC-staurosporine, FITC-NADH and FITC-NADPH in the second, which have similarfluorescence quantum yield behaviour in the studied buffers. DSSA seems to be unique fromall the studied buffers due to the presence of two dyes in its structure.Keywords: Hofmeister series, Fluorescent probes, Kosmotropes, Chaotropes, Fluorescencequantum yield.

Introduction

Fluorescent compounds are the major class of compounds used in protein-ligand interaction studies,ligand binding assays, cell labelling agents and as protein biomarkers1-3. There are a number of recentapplications of probes as chemical proteomic tools making them part of the biochemist tool box. The propertiesof the fluorescent compounds are dependent on pH of the medium, temperature, refractive index, extinctioncoefficient of compounds etc.4 The effects of these traditional parameters are widely studied in thephotochemistry perspective. Inherently, macromolecules are subjected to Hofmeister series experiments todetermine the appropriate protein precipitating conditions5. The protein precipitating reagents like ammoniumsulphate 6, denaturing agents like urea and guanidine isothiocyanate are widely used and their effects on proteinaggregation and aqueous environment distribution are well studied7-10. At molecular level, the currentunderstanding of the Hofmeister series effects on proteins is due to either effects of ions on water structure ordue to salt protein binding. The water structure hypothesis states that some ions (“kosmotropes”) enhance the

International Journal of ChemTech Research CODEN (USA): IJCRGG ISSN: 0974-4290

Vol.8, No.12 pp 348-359, 2015

Phani Kumar Pullela et al /Int.J. ChemTech Res. 2015,8(12),pp 348-359. 349

structure of the surrounding ions and enhance the hydrophobic effect, thereby stabilizing the proteins and someions (“chaotropes”) disrupt the water structure and denature the proteins11-13. The second salt-protein hypothesisstates that ions which denature proteins are the ones which bind them and the stabilizing salts are excluded fromthe protein surface. Recently, Shimizu et al. reported Kirkwood-Buff theory based calculations and foundevidence in support of salt-protein hypothesis14. Though there are theoretical models to explain Hofmeisterseries, the only experimental protein studied to date is bovine serum albumin (BSA) 15 and often the Hofmeisterseries results with other proteins are complicated and do not obey Hofmeister series. The Hofmeister series wasobserved in a wide range of phenomena including colloidal chemistry, membrane effects, surface and polymerchemistry16-20. Interestingly, the fluorescent dyes like proteins also have varying hydrophobic characteristicsand show aggregation and precipitation in aqueous solutions. It is interesting to understand the effects ofHofmeister salts and BSA on fluorescent dyes considering the similarity in hydrophobicity and aggregationproperties. It is common for a biochemist to observe that the assay conditions often change fluorescentbehaviour of dyes drastically and these changes could be mistaken for unique results. There is no systematicstudy of these Hofmeister salts on small molecule fluorescent dyes. Considering the increase in chemicalproteomic applications of fluorescent probes in biochemistry, characterization of fluorescent properties inbiochemical assay conditions is essential. In this paper we discuss the effect of Hofmeister salts on fluorescentdyes and show that dyes are different from macromolecules, and have unique behaviour with Hofmeister saltsand BSA.

Materials and Methods

All salts, buffers, and enzymes, except where noted, were from Sigma-Aldrich and were of biochemicalreagent grade. They were used without any further treatment or purification. Buffer pH was adjusted usingeither NaOH or HCl. Standard sample of BSA (1 mg/mL) was obtained from Amersham Biosciences anddiluted to 0.1 mg/mL in 100mM HEPES buffer. Fluorescein, FITC, HEPES base, urea, sodium chloride,ammonium sulphate, guanidine isothiocyanate, and sodium hydroxide were purchased from Aldrich. Thebuffers used in this study are 100mM HEPES and 100mM HEPES containing 1M Hofmeister salt adjusted topH 7.4. Fluorescence measurements were made on a Thermo Appliskan Multimode Microplate Reader at 25°C,using 96-well plates. The reader was equipped with 485nm excitation and 520-nm emission filters, both with a10nm band pass. A Beckman-Coulter DU 700 UV/VIS spectrophotometer was used for analysis of columnfractions, as well as for the UV/Vis-based kinase assay measurements. All spectroscopic measurements weretaken at 25oC in a 1 mL quartz cuvette.

Results and Discussion

Selection of Fluorescent compounds

Our group is involved in development of fluorescent analogues for different biochemical applicationsincluding cell permeable probes for quantitation of changes in the cellular thiols (DSSA) 21, estradiol analoguewith a three carbon amine linker at 17 α-position (E2-FITC) as zebrafish estrogen receptor probe22,23. As areference, fluorescein and FITC were used along with NADH-FITC (FITC attached to NADH), NADPH-FITC(FITC attached to NADPH), and staurosporine-FITC (FITC attached to staurosporine). All the compoundsselected for this study have common fluorescein as core molecules and allow the comparison betweenmolecules. DSSA has a cyclized rhodamine and FITC attached on either side of cystamine. DSSA probes areproven to cross the bacterial cell membranes, offering molecules with molecular weight close to 1000 and havea hydrophobic core with a protruding acid group providing a micellar type molecule21, 28. E2-FITC has anestrogen core attached to FITC and a phenolic group on steroidal aromatic ring. The secondary alcohol onsteroidal D-ring and acid group on FITC could act as a hydrophobic sphere with polar groups on the surface.NADH-FITC and NADPH-FITC are in the molecular weight range of 600 and offer low molecular weightmimics of biochemical molecules and on the other hand staurosporine-FITC is a high molecular weight mimicin the molecular weight range of 900. All the probes selected were assumed to form hydrophobic core withpolar group on the surface similar to proteins and the concentrations at which this work is performed is farbelow the possible aggregate-forming concentrations of respective dyes.


DSSA

O

N

O

N(Et)2(Et)2N

S NH

S

S

NH

O OHO

COOH

E2-FITC

HO

H

H CH3 OH

HN

NH

S

O

HO

OHOOC

H

FITC-Staurosporine

NN

HN O

OH H

O

HN

S

HN

O

COOH

OH

O

FITC-NADPH

N

H2N

O

O

OHHO

OP

O

HO

O

P

O

OH

O

N

N

N

N

HN

O

HOPO

HO

OH

S

HN

O

COOH

OH

O

O


FITC-NADH

NH2N

O

O

OHHO

OP

O

HO

O

P

O

OH

O

N

N

N

N

HN

OH

HO

S

HN

O

COOH

OH

O

O

Aqueous solutions of fluorescent compounds

FITC and fluorescein have solubility in water and the stocks were made accordingly in pH 7.4, 100mMHEPES buffer. Other five fluorescent compound’s stocks are prepared either in methanol or DMSO and dilutedto working concentrations in pH 7.4, 100mM HEPES buffer. In all the working solutions, the organic solventconcentration was maintained below 0.1%. As a note, pH of the buffer is always measured before and afterdissolving fluorescent compound and if needed pH was adjusted to 7.4. Similarly, after addition of 1MHofmeister series salts to the HEPES buffer, pH was adjusted back to 7.4.

Absorbance readings

Table 1 - A comparison of absorbance data for various fluorescein based dyes.

Probe HEPES BSA Urea (NH4)2SO4 NaCl GuanidineIsothiocyanate

Fluorescein 0.485 0.413 0.391 0.487 0.458 0.408FITC 0.546 0.511 0.394 0.597 0.592 0.497FITC-NADH 0.430 0.469 0.669 0.808 0.596 0.578FITC-NADPH 0.208 0.306 0.331 0.484 0.411 0.306E2-FITC 0.409 0.420 0.361 0.482 0.434 0.499FITC-staurosporine 0.143 0.182 0.271 0.350 0.267 0.188DSSA 0.010 0.034 0.032 0.036 0.027 0.024DSSA (reduced) 0.030 0.026 0.025 0.044 0.034 0.045

Absorbance readings were recorded for all seven fluorescent compounds at 485nm. The concentrationsused for the measurement were 1.28µM, 2.56µM, 5.12µM and 10.24µM in quadruplicates Table 1. Theseconcentrations were chosen in such a way that the absorbance values are within the range of 0.01 to 1.0absorbance units. The concentrations at which the linear behaviour is observed are taken as appropriateconcentrations for fluorescent studies. The concentration- dependent linear behaviour of absorbance is shown inFigure 1. DSSA has random absorbance at 485nm and only above these concentrations, the absorbance isconcentration-dependent and is shown in Figure 2. DSSA probes have cyclized rhodamine which was reportedby us to possess ability to quench FITC absorbance and fluorescence. It is the reason for requirement of higherconcentrations of DSSA probes compared to other FITC probes.


Figure 1 - Absorbance of different fluorescent probes at 485nM measured at different concentrations1.28μM, 2.56μM, 5.12μM and 10.24μM. The buffer used for experiment is 100Mm HEPES, pH-7.4 andthe UV-Visible Spectrophotometer is Beckmann-Coulter DU700.

Figure 2 - Absorbance of DSSA measured with increasing concentrations of dye.

Fluorescence readings

Fluorescence readings were recorded for all seven fluorescent compounds at 485nm.The concentrationused for the measurement were 1.28µM, 2.56µM, 5.12µM and 10.24µM in triplicates Table 2. The fluorescencevalues measured from the Appliskan multimode microplate reader and with the appropriate buffer controls areapplied in a single 96 well plate. The fluorescent intensity values are referenced to fluorescein's intensity andare not absolute numbers like absorbance values. Considering the fluorescent quantum yields calculated arerelative to fluorescein, this referencing to fluorescein gave reproducible and reliable results. The concentration-dependent linear behaviour of fluorescence intensity of all seven compounds is shown in Figure 3. DSSA probehas random fluorescence at 520nM and only above these concentrations; the fluorescence is concentration-dependent and is shown in Figure 4.

Table 2 - Fluorescence measurements


Fluorescein 46859 36442 49904 52221 59839 6709FITC 27728 29658 35723 33588 40738 3773FITC-NADH 27376 32449 38183 29305 39115 6641FITC-NADPH 16865 18895 22780 22951 28124 3115E2-FITC 7505 8842 8335 6993 9507 3190FITC-staurosporine 7133 7880 9780 9141 11427 1569DSSA 242 250 301 290 348 153DSSA (reduced) 936 996 1327 1288 1557 353


Figure 3 - The variation of fluorescence yield with absorbance for dyes in the micro molar range ofconcentration.

0

50

100

150

200

250

300

0 2 4 6 8 10 12

Fl. i

nten

sity

Concentration (μM)

Figure 4 - The change in fluorescence with increasing DSSA concentration.

Fluorescence quantum yield measurement

The literature reported quantum yield for fluorescein in 0.1 N NaOH solution is 0.92 25. We used thisreference value and calculated fluorescence quantum yield for fluorescein at pH 7.4, 100mM HEPES buffer, forthe same buffer containing 1 M Hofmeister series salts and also 0.1 mg/mL BSA Table 3. Using thesefluorescein quantum yields, we calculated the fluorescent quantum yields of other six compounds under exactsame conditions Table 4. All the fluorescence readings are referenced to fluorescein (standard) and this way thequantum yields calculated are more reliable and accurate. The following formula is used to calculate thefluorescence quantum yields and each reported quantum yield is the average of twelve individual readings(triplicates of four different concentrations). The relative quantum yield 4 is generally determined by comparingthe wavelength-integrated intensity of an unknown sample to that of a standard. The quantum yield of theunknown sample is calculated using:

Where Q is the quantum yield, I is the integrated intensity, η is the refractive index, and OD is the opticaldensity. The subscript R refers to the reference fluorophore of known quantum yield.


Table 3 - Quantum yields of fluorescein under different buffer conditions

HEPES BSA Urea (NH4)2SO4 NaCl GuanidineIsothiocyanate

Quantum yield 0.730 0.667 1.024 0.762 0.887 0.097Standard deviation 0.020 0.020 0.034 0.024 0.028 0.003

Table 4 - A summary of the calculated quantum yields for all the fluorescein based dyes.


Fluorescein 0.73 0.68 1.02 0.76 0.89 0.10FITC 0.38 0.44 0.73 0.40 0.47 0.04FITC-NADH 0.48 0.53 0.49 0.24 0.42 0.07FITC-NADPH 0.61 0.46 0.58 0.38 0.45 0.07E2-FITC 0.14 0.16 0.19 0.10 0.15 0.03FITC-staurosporine 0.38 0.33 0.29 0.19 0.29 0.05DSSA 0.18 0.06 0.08 0.06 0.09 0.04DSSA (reduced) 0.24 0.29 0.42 0.21 0.31 0.05

Refractive index

We used Bausch & Lomb Abbe-3L refractometer at 25oC using sodium D line at 589 nm (ηD25) to

obtain refractive index of buffers as shown in Table 5. To determine the accuracy of our measurement andproper functioning of the refractometer, a standard compound’s refractive index is determined and comparedwith the literature published value.

Table 5 - Refractive index for buffers used in this study.

Buffer conditions HEPES BSA Urea (NH4)2SO4 NaCl GuanidineIsothiocyanate

Refractive index 1.336 1.334 1.352 1.370 1.354 1.397

Discussion

The relationship between fluorescence and absorbance

The relationship between fluorescence and absorbance at increasing concentrations is expected to be alinear one. Non-linearity may point to the existence of significant concentration dependent physico-chemicalinteractions such as pi-pi interactions, self-aggregation or dye plastic adhesion. Depending on the specificnature, magnitude of interaction, fluorescence, absorbance or both will be affected. The various plots Figure 5of fluorescence against absorbance are almost linear. This shows that there is no evidence of any significantamount of the above mentioned concentration-dependent effects.

Fluorescence yield as a function of dye concentration

The fluorescence yields were calculated using the equation

Q - Quantum yield, I - integrated intensity, η - refractive index, OD - optical density. The subscript R refers tothe reference.


The yield appears to be steady beyond 2µM, possibly emphasizing the existence of a concentrationthreshold beyond which dye-plastic or in general dye-container interactions can be neglected. In qualitativeterms, the variation shown by the fluorescence yield on the higher concentration end follows hydrophobicitytrends. It is noted that the order of fluorescence yields is: FITC, NADPH-FITC, NADH-FITC > staurosporine-FITC > DSSA, E2-FITC.

Earlier discussions have already pointed to the existence of some hydrophobic interactions in DSSAprobes 28. The same can be said of E2-FITC given the relatively large size and hence largely hydrophobic natureof the E2 steroidal core. Staurosporine-FITC has an intermediate fluorescence yield despite the large size of thestaurosporine molecule. A closer look at its structure suggests this sugar-based protein kinase inhibitor iscapable of hydrogen bonding, lessening the extent of hydrophobic interactions. The balance of hydrogenbonding to solvent vs. hydrophobic interactions dictates the differences shown in the behaviour of staurosporineand E2-containing FITC dyes. Lastly, FITC, NADPH-FITC and NADH-FITC being the least hydrophobic,exhibit the highest fluorescence yields.

Figure 5 - The variation of fluorescence with absorbance for various FITC-containing dyes.

The effects of lyotropic salts on dye fluorescence yields

The Figure 6 shows concentration ranges for which quantum yield is derived. At low dyeconcentrations, the effect of dye container interactions becomes significant and hence the lower concentrationshave variation in quantum yields. Towards higher concentrations, this trend is not observed and the quantumyield is independent of concentration of dye. Figure 7 shows the effects of NaCl, (NH4)2SO4, urea, BSA andguanidine isothiocyanate on the quantum yield of various FITC-containing dyes. Irrespective of the dye, thetrend in fluorescence characteristics of each Hofmeister salt on the dyes (relative to one another) is sameindicating that the fluorescein core responds same way to each salt. This suggests that different compoundscould have differential fluorescence in different media but for the same fluorescence core, the effects remainsame. Conjugation of FITC on a small molecule changes the fluorescence intensity, quantum yield etc. of thatcompound, but in qualitative terms fluorescein ring, which is responsible for fluorescence from the moleculefeels same effect from Hofmeister salts. It is noteworthy that in the dye series investigated, by monitoring thefluorescein core fluorescence, DSSA is the only one with a donor-quencher pair 28. It should be noted thatDSSA will generally not be in alignment with most of the discussed trends. This molecule has significantlylower fluorescence yield compared to other molecules, but the affect of Hofmeister series salts is inline withother compounds in qualitative terms. The cyclized Rh-B in DSSA 28 is known to affect both absorbance andfluorescence of FITC and the same is reflected in presence of the lyotropic salts.


Figure 6 - The variation of quantum yield with dye concentration at pH 8.

Figure 7: Effects of various additives on the fluorescence yield of FITC-based dye.


Hofmeister series salts & fluorescein compounds

The effects of NaCl and (NH4)2SO4, both of which give relatively high quantum yield, follow someconsistent pattern whereby the latter diminishes the quantum yield to a greater extent. According to theHofmeister series theory25 where NH4

+>Na+ and SO42->Cl-, a distinct difference should be observed in quantum

yield, instead only minimal differences are observed. However, this is consistent with the fact that in equimolaramounts, (NH4)2SO4 has a higher ionic strength, a physical property that tends to force aggregation ofhydrophobic substances. The generally high quantum yield in the presence of both salts clearly shows that at1M concentration, the fluorescein dye conjugates do not cause any appreciable salting out. The quantum yieldsin the presence of urea and BSA generally leads to high quantum yields while the solubilisation effects of ureaand BSA are noted. BSA is effective at solubilizing higher molecular weight hydrophobic molecules, likeproteins, while urea is effective for both large and small molecules. This may explain the small difference inquantum yields observed, which are generally in favour of urea. Guanidine isothiocyanate and urea both beingstrong denaturants 26 however exhibit opposite effects on the quantum yield of dyes. Guanidine isothiocyanate,a strong, charged denaturant capable of very strong associations with the dye molecules, causes dramaticreductions in all the FITC-based dyes studied. This marked difference, which sets it apart from even urea,another denaturant could be due to its positive charge. Studies involving the effects of denaturants onhydrophobic aromatic compounds have proved the existence of cation-pi electron system interactions 27. Theseinteractions in the case of guanidinium ion could be responsible for the decreased quantum yields.

Conclusions

The conclusions from this study are

a) The two strong protein denaturants, guanidine isothiocyanate and urea, have opposite effects on thequantum yield indicating these dyes are completely different from macromolecules like proteins whichoften obey Hofmeister series.

b) These dyes does not obey Hofmeister series, the effects on quantum yield may not be due to hydrophobiceffect or change in water structure around dyes. The non- obeying of Hofmeister series by dyes and theirunique effects on quantum yield based on the structure of the fluorescent dyes makes them a special classto study. This information will be invaluable for biochemists and performing the simple comparativefluorescent experiments in presence of salts of interest could help to address the common anomalies in theexperiments like unusual increase or decay in fluorescence, differences in quantum yields due to presenceof proteins/salts, inconsistency and differences in in vitro and in vivo assay results etc.

Acknowledgement

Authors are very thankful to Professor. Daniel S. Sem, Concordia University Wisconsin, for the originalresearch idea and his support.

Abbreviation:

FITC - Fluorescein-5-isothiocyanate, DMSO - Dimethyl sulfoxide, NADH - Nicotinamide adeninedinucleotide, NADPH - Nicotinamide adenine dinucleotide phosphate-oxidase, DSSA – Donor-disulfide-acceptor, E2-FITC - FITC attached estrogen, FITC-NADH - FITC attached NADH, FITC-NADPH - FITCattached NADPH, BSA - Bovine serum albumin, FITC-Staurosporine - FITC attached staurosporine.

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